of Shigella with Bifidobacteria

Transcription

1 Bifidobacteria Microflora Vol. 5(1), 51-55, 1986 Interaction of Shigella with Bifidobacteria Noboru OKAMURA,1* Rintaro NAKAYA,1 Hajime YOKOTA,2 Nobuya YANAI2 and Takuji KAWASHIMA2 Department of Microbiology, Tokyo Medical and Dental University School of Medicine, Yushima, Bunkyo-ku, Tokyo 113,1 and Institute of Biological Scienceș Morinaga Milk Industries, Meguro, Meguro-ku, Tokyo 1532 (Received 28 August, 1985) We examined the effects of normal components of intestinal microflora on Shigella infection, using tissue culture infection assay. A Bifidobacterium infantis strain, both viable cells and culture supernatant, interfered strongly with the invasion and/or intracellular multiplication of Shigella organismṣ Viable cells of Escherichia coli, Streptococcus faecalis, and Bifidobacterium breve also reduced, to a lesser degree, the percentage of infected HeLa or Henle 407 cells by Shigella, but those of Bacteroides fragilis had no inhibitory effect. Key words: Intestinal microflora; bacillary dysentery; laboratory model of dysentery; infection of tissue culture cells; Bifidobacterium infantis; Shigella flexneri Bacillary dysentery is a disease in which Shigella organisms invade and proliferate in the intestinal mucosa and induce an inflammatory response. To manifest pathogenicity, Shigella must (i) possess smooth LPS O antigen, (ii) have the ability to invade epithelial cells and proliferate therein, and (iii) elaborate a toxin after cell invasion (1, 3). Studies carried out by Formal et al. (1) and by others (5, 9, 10) showed that epithelial cell invasiveness of Shigella spp. is a prerequisite of the disease. Several laboratory models have been used to assess the virulence of Shigella and other invasive enteric organisms and to study the mechanisms of their pathogenesis. Virulent strains of Shigella can infect cultured epithelial cells in vitro, cause purulent keratoconjunctivitis in guinea pigs after inoculation into the conjunctival sac (Sereny test), and manifest symptoms of dysentery in starved, opiated guinea pigs, and in monkeys after oral challenge. Shigella strains isolated from patients and carriers have been shown to be exclusively virulent judged on the basis of positive tests in the above experimental models. In contrast, mutants that lost epithelial cell invasiveness are exclusively negative in these laboratory models, indicating that they are avirulent (1, 3, 5). On the other hand, hosts provide themselves several defense mechanisms at the intestinal mucosal surfaces to counter bacterial infections (2, 4). Normal components of intestinal microbial flora have long been considered to be one of the candidates of host defense mechanisms at the mucosal surfaces, providing environments unfavorable for the invading microorganisms to colonize in the gut mucosa (7, 12). We have recently done several experiments, using tissue culture infection assay, to examine the effects of bacterial species, especially Bifidobacterium spp., in the intestinal flora, on the invasion of Shigella. 1. Inhibitory Effect of B. infantis on Tissue Culture Cell Infection by S. flexneri S. flexneri was used throughout

2 N. OKAMURA et al. Fig. 1. Inhibitory effect of B. infantis MDI-15 on HeLa cell infection by S. flexneri HeLa cell monolayers were mixed with (A) S. flexneri alone (control) ; (B) S. flexneri and B. infantis (mixed simultaneously) ; (C) S. flexneri and B. infantis (S. flexneri was allowed to infect 2 hr after addition of B. infantis); (D) S. flexneri and B. infantis (B. infantis was added to the monolayers, incubated for 2 hr, and removed by washing, and then S. flexneri was allowed to infect). Fig. 2. Effect of the number of viable cells of B. infantis MDI-15 on the frequency of HeLa cell infection by S. flexneri Abscissa indicates log number of viable cells of B. infantis per chamber (0.5 ml). When the monolayers were incubated with B. infantis for 2 hr, washed to remove the bacteria, and then infected by S. flexneri, the infection frequency was slightly increased (Fig. 1). These experiments showed that bifidobacteria mixed with cultured cells interfered with the invasion and/or intracellular multiplication of Shigella organisms. The change in the frequency of cell infection the study. This strain is a rough mutant of S. flexneri 2a Although this strain does not produce keratoconjunctivitis in guinea pigs, it invades tissue culture by S. flexneri was examined by mixing various numbers of viable cells of B. infantis with the S. flexneri-cell infection system. cells and proliferates therein (10). HeLa- The experiments were performed by the S3 and Intestine 407 cell monolayers were simultaneous mixing of S. flexneri and B. used to study the virulence of the Shigella infantis with the cell monolayers (Fig. 2). organisms as an in vitro assay system. The The frequency of cell infection was decreased procedure for cell culture, infection of in proportion to the number of viable cells monolayers by Shigella organisms, and determination of B. infantis added. The inhibition of cell of cell infection frequency by infection by B. infantis was no longer apparent Shigella were the same as reported previously when the number of viable cells was decreased (6, 9, 10). Portions of the broth culture of to the order of 105 per chamber. B. infantis MDI-15 (107 organisms/chamber) The following experiments were then were added to the chambers of HeLa cell monolayers infected with S. flexneri undertaken to rule out the possibility that addition of Bifidobacterium induced a change under various conditions. As shown in in ph and reduced a viable count of Shigella Fig. 1, the frequency of infection of HeLa organisms in the mixture. Changes in ph cells by S. flexneri was much less when S. flexneri and B. infantis were mixed simultaneously into the monolayers than when only values and viable counts were determined, when a mixed culture of S. flexneri and B. infantis MDI-15 in MEM supplemented S. flexneri was added. Almost the same inhibitory with 10% calf serum was incubated effect was observed when S. flexneri was infected 2 hr after addition of B. infantis. at 37 C for 5 hr in a 10% CO2 incubator. As controls, individual strains were incubated

3 SHIGELLA AND BIFIDOBACTERIA 53 Fig. 3. Changes in ph of medium and viable count when S. flexneri and B. infantis MDI-15 were grown in mixture. (A) S. flexneri alone; œ- œ,ph; œ-- œ, CFU: (B) B. infantis MDI-15 alone; -, ph: --, CFU: (C) Mixed culture of S. flexneri and B. infantis MDI-15; -, ph; œ-- œ, CFU of S. flexneri; --, of B. infantis. Table 1. Effect of various bacterial strains on HeLa cell infection by S. flexneria a Bacterial suspensions of S. flexneri (5 ~ 107/0.1 ml) and the strain indicated (5 ~ 107/0.1 ml) were mixed with the HeLa cell monolayers (0.3 ml), and incubated at 37 C for 5 hr. b Number of infected cells/total number of cells observed. Infection frequency relative to that when S. flexneri alone c was the infecting agent. alone. We found that both strains in the mixed culture gave a change in ph value and viable count comparable to that of the single culture (Fig. 3). 2. Effect of Various Bacterial Strains on Tissue Culture Cell Infection by S. flexneri We then examined several bacterial strains belonging to the common species of human intestinal microflora for their inhibitory activity to cell infection by Shigella. As shown in Table 1, strains of Bifidobacterium spp., E. coli, and Streptococcus faecalis except Bacteroides fragilis all exhibited an inhibitory effect on the cell infection by Shigella, though not as strongly as B. infantis MDI Inhibitory Effect of Culture Supernatant of B. infantis on Cell Infection by S. flexneri In the next step of the study, the culture

4 54 N. OKAMURA et al. Fig. 4. Inhibitory effect of culture supernatant of B. infantis MDI-15 on the infection of HeLa and Henle 407 cells by S. flexneri œ, HeLa cell infection;, Henle 407 cell infection; bars indicate the standard deviation of the mean obtained in three independent experiments. supernatant of B. infantis MDI-15 was tested for inhibitory activity to cell infection using HeLa and Intestine 407 cell monolayers. Aliquots (0.1 ml) of the culture supernatant of B. infantis and its GAM broth dilutions were mixed with the HeLa cell monolayers (0.3 ml) to which a suspension of S. flexneri (0.1 ml) was added simultaneously. Controls were the monolayers infected with Shigella organisms alone and those mixed with fresh GAM broth (0.1 ml) and a suspension of Shigella organisms (0.1 ml). As shown in Fig. 4, addition of the culture supernatant of B. infantis reduced the frequency of cell infection by Shigella. The culture supernatant of B. infantis MDI-15 was dialyzed against PBS and then tested for activity. The dialyzed culture supernatant also had an inhibitory effect in the infection system. These experiments proved the presence of some active factor(s) in the culture supernatant that protects the cells from invasion by and proliferation of S. flexneri organisms. Recently many articles have been published concerning the beneficial effects of Bifidobacterium, as one of the most commonly found species of human intestinal microflora, on its host (8, 11, 12). However, to the best of our knowledge, there have been few studies in which the interaction between enteric pathogens and bifidobacteria has been analyzed using laboratory models of enteric infections. The studies presented in this review and elsewhere (7) demonstrated for the first time the protective activity of Bifidobacterium and its high molecular weight products for epithelial cells from invasion and/or multiplication of Shigella. It may be reasonable to account for non-specific defense mechanisms of the intestinal mucous membrane partly by such activities of Bifidobacterium as described above. Further investigations are required to explain the mechanisms of these protective activities and the mode of modification of epithelial cells by the active factors of B. infantis. It is not known yet whether the active factors inhibit the invasion of cells by Shigella organisms or intracellular proliferation of invading organisms. In addition, it should be of interest to explore whether the protective activity as observed with E. coli and other microflora is shared by B. infantis. Isolation, purification and characterization of the infection-protective factors of B. infantis are now under study. The effectiveness of the factors in other laboratory models must also be confirmed. We believe that such research would clarify the role of Bifidobacterium in protection against enteric infections. References (1) Formal, S.B., T.L. Hale, and P. J. Sansonetti Invasive enteric pathogens. Rev. Infect. Dis. 5: S702 S707. (2) Freter, R Bacterial association with the mucus gel system of the gut, p In D. Schlessinger (ed.), Microbiology-1982, American Society for Microbiology, Washington, D.C. (3) Levine, M.M., J.B. Kaper, R.E. Black, and M.L. Clements New knowledge on pathogenesis of bacterial enteric infections as applied to vaccine development. Microbiol. Rev. 47:

5 SHIGELLA AND BIFIDOBACTERIA (4) McClelland, D.B.L Bacterial and viral infections of the gastrointestinal tract, p In P. Asquith and P.G.H. Gell (eds.), Immunology of the gastrointestinal tract, Churchill Livingstone, Edingburgh. (5) Nakaya, R Mucous membrane infections, p In T. Amano, H. Uetake and H. Fukumi (eds.), Viruses, bacteria, and their infections, Iwanami Shoten, Tokyo (in Japanese). (6) Nakaya, R., N. Okamura, M. Ogawa, N. Goto, A. Nakamura, H. Yoshikura, and H. Ogawa Drug resistance of Shigella and experimental chemotherapy for shigellosis, p In Y. Takeda and T. Miwatani (eds.), Bacterial diarrheal diseases, Martinus Nijhoff Publ., Boston, The Hague, Dordrecht, Lancaster. (7) Nakaya, R., H. Tomeoku, N. Okamura, and M. Higaki Intestinal microflora and enteric infection: inhibitory activity of Bifidobacterium on cell invasion by Shigella flexneri. J. Pediatric Practice 47: (in Japanese). (8) Nakaya, R., T. Chida, and H. Shibaoka Antimicrobial agents and intestinal microflora. Bifidobacteria Microflora 1: (9) Okamura, N., T. Nagai, R. Nakaya, S. Kondo, M. Murakami, and K. Hisatsune HeLa cell invasiveness and O antigen of Shigella flexneri as separate and prerequisite attributes of virulence to evoke keratoconjunctivitis in guinea pigs. Infect. Immun. 39: ) Okamura, N., and ( R. Nakaya Rough mutant of Shigella flexneri 2a that penetrates tissue culture cells but does not evoke keratoconjunctivitis in guinea pigs. Infect. Immun. 7: ) Rasic, ( J. L. and J.A. Kurmann Bifidobacteria and their role, Birkhauser Verlag, Basel, Boston, Stuttgart. 12) Tamura, N., ( C. Hirayama, and A. Takagi Clinical significance of fecal Bifidobacterium. Saishin-igaku 38: (in Japanese).